Estrogen Deficiency Exacerbates Traumatic Heterotopic Ossification in Mice.

Background: Traumatic heterotopic ossification (HO) is a devastating sequela of orthopedic surgeries and traumatic injuries; however, few studies have explored the effects of the estrogen-deficient state on HO formation. In the present study, we investigated the impact of estrogen deficiency on ectopic cartilage and bone formation in tendon after Achilles tenotomy in an ovariectomized mouse model. Methods: A total of 45 female C57BL/6 mice were randomly divided into three groups: sham-operated (control), estrogen depletion by ovariectomy (OVX) and OVX with 17ß-estradiol supplementation (OVX + E2), with 15 animals in each group. Three weeks after OVX, all mice were subjected to an Achilles tenotomy using a posterior midpoint approach to induce HO. At 1, 3 and 9 weeks after tenotomy, the left hind limbs were harvested for histology, immunohistochemistry and immunofluorescence evaluations. The volume of ectopic bone was assessed by micro-CT. Results: Mice in the OVX group formed more ectopic cartilage 3 weeks after tenotomy, as well as ectopic bone 9 weeks after tenotomy, compared to the control group. Estrogen deficiency resulted in more severe inflammatory infiltration at the injury sites 1 week after tenotomy, involving the recruitment of more macrophages and mast cells, as well as increasing the expressions of pro-inflammatory mediators, including IL-1ß, IL-6, and TNF-α. Moreover, the local TGF-ß/SMAD signaling pathway was dysregulated after OVX, which manifested as upregulated expressions of TGF-ß and pSMAD2/3. E2 supplementation protected against OVX-induced HO deterioration, inhibited inflammatory infiltration, and downregulated the TGF-ß/SMAD signaling pathway. Conclusion: Estrogen deficiency exacerbated HO formation in the Achilles tenotomy model. These findings might be attributable to the disturbance of the inflammatory response and the activation of TGF-ß/SMAD signaling at the injury sites during the early stages of HO development.

A targeted activatable NIR-II nanoprobe for positive visualization of anastomotic thrombosis and sensitive identification of fresh fibrinolytic thrombus.

Anastomotic thrombosis prevalently causes anastomosis failure, accompanied with ischemia and necrosis, the early diagnosis of which is restricted by inherent shortcomings of traditional imaging techniques in clinic and lack of appropriate prodromal biomarkers for thrombosis initiation. Herein, a fresh thrombus-specific molecular event, protein disulfide isomerase (PDI) is innovatively chosen as the activating factor, and a thrombosis targeting and PDI-responsive turn-on near infrared II (NIR-II) fluorescence nanoprobe is firstly developed. The supramolecular complex-based nanoprobe IR806-PDA@BSA-CREKA is fabricated by assembling NIR-II emitting cyanine derivative IR806-PDA with bovine serum albumin (BSA), which could ameliorate the stability and pharmacokinetics of the nanoprobe, addressing the contradiction in the balance of brightness and biocompatibility. The NIR-II-off nanoprobe exhibits robust turn-on NIR-II fluorescence upon PDI-specific activation, in vitro and in vivo. Of note, the constructed nanoprobe demonstrates superior photophysical stability, efficient fibrin targeting peptide-derived thrombosis binding and a maximum signal-to-background ratio (SBR) of 9.30 for anastomotic thrombosis in NIR-II fluorescent imaging. In conclusion, the exploited strategy enables positive visualized diagnosis for anastomotic thrombosis and dynamic monitoring for thrombolysis of fresh fibrinolytic thrombus, potentially contributes a novel strategy for guiding the therapeutic selection between thrombolysis and thrombectomy for thrombosis treatment in clinic.

Early diagnosis of heterotopic ossification with a NIR fluorescent probe by targeting type II collagen.

Heterotopic ossification (HO) is a devastating sequela in which the pathologic extracellular matrix of the cartilage and bone forms abnormally in soft tissues following traumatic injuries or orthopaedic surgeries. Early treatment is essential for inhibiting the progression of HO but is currently limited by the absence of sensitive and specific early diagnosis. Herein, this study exploits the enrichment of type II collagen (Col2a1) in the HO cartilage formation stage towards developing a near-infrared (NIR) probe for early HO diagnosis, namely WL-808 by conjugating a Col2a1-binding peptide (WYRGRL) and a cyanine dye (IR-808). WL-808 exhibits high specificity for targeting the cartilage in vitro and in vivo with no apparent cytotoxicity. The NIR signal of WL-808 can be detected in the HO cartilage lesions as early as 1 week after injury when micro-CT cannot show any ectopic bone formation. Moreover, the probe is rarely distributed in the normal knee articular cartilage in vivo via the intravenous administration method. Taken together, WL-808 demonstrates great potential in early HO diagnosis under NIR imaging, facilitating early HO prophylaxis and treatment in the clinic.

Bioengineered miR-34a modulates mitochondrial inner membrane protein 17 like 2 (MPV17L2) expression toward the control of cancer cell mitochondrial functions.

Genome-derived microRNAs (miRNAs or miRs) control post-transcriptional gene expression critical for various cellular processes. Recently, we have invented a novel platform technology to achieve high-yield production of fully humanized, bioengineered miRNA agents (hBERAs) for research and development. This study is aimed to produce and utilize a new biologic miR-34a-5p (or miR-34a) molecule, namely, hBERA/miR-34a, to delineate the role of miR-34a-5p in the regulation of mitochondrial functions in human carcinoma cells. Bioengineered hBERA/miR-34a was produced through in vivo fermentation production and purified by anion exchange fast protein liquid chromatography. hEBRA/miR-34a was processed to target miR-34a-5p in human osteosarcoma and lung cancer cells, as determined by selective stem-loop reverse transcription quantitative polymerase chain reaction analysis. The mitochondrial inner membrane protein MPV17 like 2 (MPV17L2) was validated as a direct target for miR-34a-5p by dual luciferase reporter assay. Western blot analysis revealed that bioengineered miR-34a-5p effectively reduced MPV17L2 protein outcomes, leading to much lower levels of respiratory chain Complex I activities and intracellular ATP that were determined with specific assay kits. Moreover, Seahorse Mito Stress Test assay was conducted, and the results showed that biologic miR-34a-5p sharply reduced cancer cell mitochondrial respiration capacity, accompanied by a remarkable increase of oxidative stress and elevated apoptotic cell death, which are manifested by greater levels of reactive oxygen species and selective apoptosis biomarkers, respectively. These results demonstrate the presence and involvement of the miR-34a-5p-MPV17L2 pathway in the control of mitochondrial functions in human carcinoma cells and support the utility of novel bioengineered miRNA molecules for functional studies.

Chidamide augment sorafenib-derived anti-tumor activities in human osteosarcoma cells lines and xenograft mouse model.

Previous studies have showed promising but short-lived activity of sorafenib in osteosarcoma treatments. Researches have suggested ameliorated sensitivity to standard dose of conventional cancer therapies in combination with histone deacetylase inhibitors (HDACis) through various mechanisms. Herein, for the first time, we exploited the synergism of combination therapies with sorafenib and chidamide, a member of HDACis, in the control of OS using human OS cell lines and OS xenograft mouse model and discussed interactive mechanisms between the two drugs. The combination therapy exerted a strong synergism in the inhibition of OS cell proliferation, meanwhile prominently induced cell apoptosis and cell cycle arrest in G0/G1 phase in OS cells with increased expression of MCL-1, decreased expression of caspase-3 and P21, along with diminished level of the overlapped protein P-ERK1/2. Furthermore, oral administration of the combined treatment led to a more optical therapeutic outcome, including lower degrees of tumoral cell proliferation, greater extent of apoptosis, along with induction of cell cycle arrest in tumor tissues, while exhibiting minimal toxicity. This study shows that the combination of sorafenib and chidamide can combat OS in a synergistic fashion and prompts the promising development of innovative combined therapeutic strategies for OS.

Peptide-based semiconducting polymer nanoparticles for osteosarcoma-targeted NIR-II fluorescence/NIR-I photoacoustic dual-model imaging and photothermal/photodynamic therapies.

BACKGROUND: The overall survival rate of osteosarcoma (OS) patients has not been improved for 30 years, and the diagnosis and treatment of OS is still a critical issue. To improve OS treatment and prognosis, novel kinds of theranostic modalities are required. Molecular optical imaging and phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), are promising strategies for cancer theranostics that exhibit high imaging sensitivity as well as favorable therapeutic efficacy with minimal side effect. In this study, semiconducting polymer nanoparticles (SPN-PT) for OS-targeted PTT/PDT are designed and prepared, using a semiconducting polymer (PCPDTBT), providing fluorescent emission in the second near-infrared window (NIR-II, 1000 - 1700 nm) and photoacoustic (PA) signal in the first near-infrared window (NIR-I, 650 - 900 nm), served as the photosensitizer, and a polyethylene glycolylated (PEGylated) peptide PT, providing targeting ability to OS. RESULTS: The results showed that SPN-PT nanoparticles significantly accelerated OS-specific cellular uptake and enhanced therapeutic efficiency of PTT and PDT effects in OS cell lines and xenograft mouse models. SPN-PT carried out significant anti-tumor activities against OS both in vitro and in vivo. CONCLUSIONS: Peptide-based semiconducting polymer nanoparticles permit efficient NIR-II fluorescence/NIR-I PA dual-modal imaging and targeted PTT/PDT for OS.

The Optimal Outcome of Suppressing Ewing Sarcoma Growth in vivo With Biocompatible Bioengineered miR-34a-5p Prodrug.

Being the second most common type of primary bone malignancy in children and adolescents, Ewing Sarcoma (ES) encounters the dilemma of low survival rate with a lack of effective treatments. As an emerging approach to combat cancer, RNA therapeutics may expand the range of druggable targets. Since the genome-derived oncolytic microRNA-34a (miR-34a) is down-regulated in ES, restoration of miR-34a-5p expression or function represents a new therapeutic strategy which is, however, limited to the use of chemically-engineered miRNA mimics. Very recently we have developed a novel bioengineering technology using a stable non-coding RNA carrier (nCAR) to achieve high-yield production of biocompatible miRNA prodrugs, which is a great addition to current tools for the assessment of RNA therapeutics. Herein, for the first time, we investigated the biochemical pharmacology of bioengineered miR-34a-5p prodrug (nCAR/miR-34a-5p) in the control of ES using human ES cells and xenograft mouse models. The bioengineered nCAR/miR-34a-5p was precisely processed to mature miR-34a-5p in ES cells and subsequently suppressed cell proliferation, attributable to the enhancement of apoptosis and induction of G2 cell cycle arrest through downregulation of SIRT-1, BCL-2 and CDK6 protein levels. Furthermore, systemic administration of nCAR/miR-34a-5p dramatically suppressed the ES xenograft tumor growth in vivo while showing biocompatibility. In addition, the antitumor effect of bioengineered nCAR/miR-34a-5p was associated with a lower degree of tumoral cell proliferation and greater extent of apoptosis. These findings demonstrate the efficacy of bioengineered miR-34a-5p prodrug for the treatment of ES and support the development of miRNA therapeutics using biocompatible bioengineered miRNA prodrugs.

Bioengineered miR-328-3p modulates GLUT1-mediated glucose uptake and metabolism to exert synergistic antiproliferative effects with chemotherapeutics.

MicroRNAs (miRNAs or miRs) are small noncoding RNAs derived from genome to control target gene expression. Recently we have developed a novel platform permitting high-yield production of bioengineered miRNA agents (BERA). This study is to produce and utilize novel fully-humanized BERA/miR-328-3p molecule (hBERA/miR-328) to delineate the role of miR-328-3p in controlling nutrient uptake essential for cell metabolism. We first demonstrated successful high-level expression of hBERA/miR-328 in bacteria and purification to high degree of homogeneity (>98%). Biologic miR-328-3p prodrug was selectively processed to miR-328-3p to suppress the growth of highly-proliferative human osteosarcoma (OS) cells. Besides glucose transporter protein type 1, gene symbol solute carrier family 2 member 1 (GLUT1/SLC2A1), we identified and verified large neutral amino acid transporter 1, gene symbol solute carrier family 7 member 5 (LAT1/SLC7A5) as a direct target for miR-328-3p. While reduction of LAT1 protein levels by miR-328-3p did not alter homeostasis of amino acids within OS cells, suppression of GLUT1 led to a significantly lower glucose uptake and decline in intracellular levels of glucose and glycolytic metabolite lactate. Moreover, combination treatment with hBERA/miR-328 and cisplatin or doxorubicin exerted a strong synergism in the inhibition of OS cell proliferation. These findings support the utility of novel bioengineered RNA molecules and establish an important role of miR-328-3p in the control of nutrient transport and homeostasis behind cancer metabolism.

Specific Small-Molecule NIR-II Fluorescence Imaging of Osteosarcoma and Lung Metastasis.

Osteosarcoma is an aggressive tumor of mesenchymal origin that is more likely to spread to the lung than others, with a major impact on patients' prognosis. The optimal imaging method that can reliably detect or exclude pulmonary metastases from osteosarcoma is still scarce. Herein, two homologous types of fluorescent probes CH1055-PEG-PT and CH1055-PEG-Affibody, which show highly promising results for targeting imaging of osteosarcoma and its lung metastasis, respectively, are designed and synthesized. It is found that the NIR-II imaging quality of CH1055-PEG-PT is far superior to that of computed tomography for the early in vivo 143B tumor imaging, and this probe-guided surgery for accurate resection of 143B tumor is further performed. The high-resolution visualization of primary and micrometastatic lung lesions of osteosarcoma by using CH1055-PEG-Affibody is also demonstrated. Therefore, the attractive imaging properties of CH1055-PEG-PT and CH1055-PEG-Affibody, including high levels of uptakes, and high spatial and temporal resolution, open up opportunities for molecular imaging and clinical translation of osteosarcoma and its lung metastasis in the unique second near-infrared window.

NIR-II Fluorescence Imaging of Skin Avulsion and Necrosis.

Skin avulsion is commonly seen in individuals exposed to heavy shearing forces. Subcutaneous tissue detachment and bone fractures usually accompany skin avulsion. Thus, the estimation of the extent of damaged tissue is very important. Currently, the viability of skin and subcutaneous tissue is determined by clinical observations, and these observations always underestimate the true extent of the avulsed skin. Herein, we synthesized an innovative probe, CH1055-GRRRDEVDK (CH1055-GK), which can specifically bind to caspase-3 so as to image skin avulsion and define necrotic regions. Our uptake and binding affinity tests in apoptotic cells and evaluation of the probe ex vivo and in vivo showed that the probe has a strong ability to bind caspase-3 in skin avulsion models and that it vividly detected the necrotic area in avulsed skins. Furthermore, the increased fluorescence intensity of the probe in the avulsed skin showed a larger affected area than that determined by clinical observations in live mice. Consequently, our results indicated that observation of the caspase-3-targeted probe CH1055-GK via NIR-II imaging allowed the clear detection of skin avulsion in subjects, indicating its potential as an imaging tool for the early diagnosis of skin avulsion and the determination of necrotic margins.

A NIR-II fluorescent probe for articular cartilage degeneration imaging and osteoarthritis detection.

Articular cartilage (AC) is a complex water-bearing tissue consisting of chondrocytes, proteoglycans, and collagen. AC degeneration, which occurs in the early stage and throughout the entire course of osteoarthritis (OA), is one of the main pathological changes of OA. However, current clinical approaches are unable to detect AC degradation during the early stage of OA. Herein, a novel NIR-II probe, CH1055-WL, was developed with an organic fluorophore (CH1055) and type II collagen-binding peptide (WYRGRL) for AC targeting and degeneration imaging. In vitro and in vivo imaging studies demonstrated that CH1055-WL specifically bound to AC and permitted sensitive detection of age-related or surgically induced AC degeneration in living mice. In vitro imaging of cartilage samples from pig knee joint and in vivo imaging of live mice with the probe administered via local injection in joint cavities demonstrated that CH1055-WL specifically and efficiently bound to AC. Further evaluation of CH1055-WL revealed sensitive detection of age-related AC degeneration and surgically induced AC degeneration in living mice. Our results indicated that the cartilage-targeting probe CH1055-WL allowed visual monitoring of AC degeneration in living subjects, thus displaying promise for early OA detection.

Comparative effectiveness study between negative pressure wound therapy and conventional wound dressing on perforator flap at the Chinese tertiary referral teaching hospital.

BACKGROUND: Perforator flap techniques with conventional wound dressing have being extensively used in the management of soft-tissue defects. However; the flap's survival rate is not always guaranteed and the wound healing time always long. The aim of this study was to investigate the clinical effectiveness use of a freshly transplanted perforator flap in conjunction with Vacuum-assisted closure (VAC) for better clinical outcomes. METHODS: A prospective, randomized, effectiveness study comparing the clinical outcomes of VAC versus traditional wrap and bandages for the treatment of open wounds that required hospital admission and operative debridement using perforator flaps, was carried out from March 1, 2014 to March 31, 2016 at Wuhan University Zhongnan Hospital. Fifty-one eligible patients were randomized into two groups; study group (perforator flaps covered by VAC) and control group (perforator flaps covered by traditional wrap and bandages). The measured clinical endpoints included the time of the first post-operative dressing change, pain visual analogical scale, perforator flap infection rate, 95% perforator flap healing time and percentage of survived perforator flap. RESULTS: There was no statistically significant difference in the demographic profiles in the two cohorts. There were statistically significant differences in the clinical endpoints in the two groups (p < 0.001; p < 0.05, Table 2). CONCLUSIONS: In summary, VAC combining with perforator flap technique, can diminish accumulated exudation of the transferring flap, protect against postoperative infection, prolong the interval between perforator flap relocation and first postoperative dressing change, decrease pain during removal of dressing, increase perforator flap survival rate, and shorten wound healing time, with a good aesthetic outcome, a good mobility and a satisfactory therapeutic result.

HIF-1α inhibits IDH-1 expression in osteosarcoma.

Recently, hypoxia inducible factor-1 (HIF-1) was reported to be correlated with isocitrate dehydrogenase 1 (IDH-1) in several types of tumors. However, the expression and significance of HIF-1 and IDH-1 in osteosarcoma is still unknown. In the present study, the expression levels of IDH-1 and HIF-1α in 35 formalin-fixed paraffin-embedded sections from osteosarcoma patients were investigated by immunohistochemistry. The expression levels of IDH-1 and HIF-1α in human osteosarcoma cell lines (MG-63 and 143B) were further detected by western blotting under normal and hypoxic conditions. In addition, HIF-1α was downregulated via lentiviral vector­mediated RNA interference (RNAi) in the MG-63 human osteosarcoma cell line. The results revealed that HIF-1α was negatively correlated with IDH-1 in the osteosarcoma tissues. Both in MG-63 and 143B cell lines, the expression of HIF-1α was increased while IDH-1 was decreased under a hypoxic condition compared to normal conditions. HIF-1α downregulation promoted IDH-1 expression in the MG-63 cell line under either normal or hypoxic conditions. In conclusion, our findings suggest that HIF-1α inhibits IDH-1 in osteosarcoma and consequently increases the incidence of osteosarcoma.

Downregulation of IDH2 exacerbates the malignant progression of osteosarcoma cells via increased NF-κB and MMP-9 activation.

Isocitrate dehydrogenase 2 (IDH2) is a mitochondrial NADP-dependent isocitrate dehydrogenase. It is considered to be a novel tumor suppressor in several types of tumors. However, the role and related mechanism of IDH2 in osteosarcoma remain unknown. The expression and significance of IDH2 were investigated by immunohistochemistry in formalin-fixed paraffin sections from 44 osteosarcoma patients. IDH2 was downregulated via lentiviral vector­mediated RNA interference (RNAi) in the Saos-2 and MG-63 human osteosarcoma cell lines. The effect of IDH2 downregulation on human osteosarcoma was studied in vitro by MTT, flow cytometry and invasion assays. Nuclear factor-κB (NF-κB) and matrix metalloproteinase-9 (MMP-9) assays were also used to study the likely molecular mechanism of IDH2 downregulation on the malignant progression of osteosarcoma cells. The results revealed that the expression of IDH2 was inversely correlated with pathological grade and metastasis in osteosarcoma. IDH2 downregulation promoted a pro-proliferative effect on the Saos-2 and MG-63 osteosarcoma cell lines. IDH2 downregulation accelerated cell cycle progression from S to G2/M phase. The pro-proliferative effect induced by IDH2 downregulation may be ascribed to increased NF-κB activity via IκBα phosphorylation. The invasive activity of osteosarcoma cells was also significantly promoted by IDH2 downregulation and may result from elevated MMP-9 activity. In conclusion, IDH2 downregulation may exacerbate malignant progression via increased NF-κB and MMP-9 activity and may implicate the potential biological importance of IDH2 targeting in osteosarcoma cells. Downregulation of IDH2 exacerbates the malignant progression of osteosarcoma cells via increased NF-κB and MMP-9 activation.

The iron chelator Dp44mT suppresses osteosarcoma's proliferation, invasion and migration: in vitro and in vivo.

Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), the novel iron chelator, has been reported to inhibit the tumorigenesis and progression of various cancer cells, including neuroblastoma, neuroepithelioma and prostate cancer. However, whether Dp44mT has anticancer effects in osteosarcoma is still unknown. Here, we investigated the antitumor action of Dp44mT in osteosarcoma and its underlying mechanisms. A human osteosarcoma 143B cell line in vitro and 143B xenograft in nude mice in vivo were utilized, the anticancer effects of Dp44mT were examined through methods of MTT assay, transwell, wound healing assay, flow cytometry, western blot, immunohistochemistry and H&E staining. We showed that Dp44mT inhibits cell proliferation, invasion and migration in vitro. In addition, flow cytometry further illustrated that Dp44mT suppression of 143B cell proliferation, invasion and migration were partially due to induction of cell apoptosis, cell cycle arrest in S phase and ROS production. Also in vitro and in vivo, the expression levels of Bcl2, Bax, Caspase3, Caspase9, LC3-II, ß-catenin and its downstream targets such as C-myc and Cyclin D1 demonstrated that cell apoptosis and autophagy, as well as Wnt/ß-catenin pathway were involved in Dp44mT induced osteosarcoma suppression. The Dp44mT inhibition of osteosarcoma was further verified via animal models. The findings indicated that in vivo Dp44mT showed a significant reduction in the 143B xenograft tumor growth and metastasis. In conclusion, our data demonstrated that Dp44mT has effective anticancer capability in osteosarcoma and that may represent a promising treatment strategy for osteosarcoma.